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J Bacteriol. 2010 Mar;192(5):1387-94. doi: 10.1128/JB.01423-09. Epub 2010 Jan 4.

ATP-dependent carboxylation of acetophenone by a novel type of carboxylase.

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  • 1Philipps-University Marburg, Karl-von-Frisch-Str. 8, D-35043 Marburg, Germany.


Anaerobic ethylbenzene metabolism in the betaproteobacterium Aromatoleum aromaticum is initiated by anaerobic oxidation to acetophenone via (S)-1-phenylethanol. The subsequent carboxylation of acetophenone to benzoylacetate is catalyzed by an acetophenone-induced enzyme, which has been purified and studied. The same enzyme is involved in acetophenone metabolism in the absence of ethylbenzene. Acetophenone carboxylase consists of five subunits with molecular masses of 70, 15, 87, 75, and 34 kDa, whose genes (apcABCDE) form an apparent operon. The enzyme is synthesized at high levels in cells grown on ethylbenzene or acetophenone, but not in cells grown on benzoate. During purification, acetophenone carboxylase dissociates into inactive subcomplexes consisting of the 70-, 15-, 87-, and 75-kDa subunits (apcABCD gene products) and the 34-kDa subunit (apcE gene product), respectively. Acetophenone carboxylase activity was restored by mixing the purified subcomplexes. The enzyme contains 1 Zn(2+) ion per alphabetagammadelta core complex and is dependent on the presence of Mg(2+) or Mn(2+). In spite of the presence of Zn in the enzyme, it is strongly inhibited by Zn(2+) ions. Carboxylation of acetophenone is dependent on ATP hydrolysis to ADP and P(i), exhibiting a stoichiometry of 2 mol ATP per mol acetophenone carboxylated. The enzyme shows uncoupled ATPase activity with either bicarbonate or acetophenone in the absence of the second substrate. These observations indicate that both substrates may be phosphorylated, which is consistent with isotope exchange activity observed with deuterated acetophenone and inhibition by carbamoylphosphate, a structural analogue of carboxyphosphate. A potential mechanism of ATP-dependent acetophenone carboxylation is suggested.

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